Apoptosis is an active and programmed physiological process for eliminating superfluous, altered or malignant cells (Earnshaw, 1995; Duke et al., 1996). The terms “transformed” and “tumorigenic” will be used interchangeably herein. Apoptosis is characterized by shrinkage of cells, segmentation of the nucleus, and condensation and cleavage of DNA into domain-sized fragments, in most cells followed by internucleosomal degradation. The apoptotic cells fragment into membrane-enclosed apoptotic bodies. Finally, neighboring cells and/or macrophages will rapidly phagocytose these dying cells (Wyllie et al., 1980; White, 1996). Cells grown under tissue-culture conditions and cells from tissue material can be analyzed for being apoptotic with DNA-staining agents, such as, e.g., DAPI, which stains normal DNA strongly and regularly, whereas apoptotic DNA is stained weakly and/or irregularly (Noteborn et al., 1994; Telford et al., 1992).
The apoptotic process can be initiated by a variety of regulatory stimuli (Wyllie, 1995; White 1996; Levine, 1997). Changes in the cell survival rate play an important role in human pathogenesis of diseases, e.g., in cancer development and autoimmune diseases, where enhanced proliferation or decreased cell death (Kerr et al., 1994; Paulovich, 1997) is observed. A variety of chemotherapeutic compounds and radiation have been demonstrated to induce apoptosis in tumor cells, in many instances, via wild-type p53 protein (Thompson, 1995; Bellamy et al., 1995; Steller, 1995; McDonell et al., 1995).
Many tumors, however, acquire a mutation in p53 during their development, often correlating with poor response to cancer therapy. Certain transforming genes of tumorigenic DNA viruses can inactivate p53 by directly binding to it (Teodoro, 1997). An example of such an agent is the large T antigen of the tumor DNA virus SV40. For several (leukemic) tumors, a high expression level of the proto-oncogene Bcl-2 or Bcr-ab1 is associated with a strong resistance to various apoptosis-inducing chemotherapeutic agents (Hockenberry, 1994; Sachs and Lotem, 1997).
For such tumors lacking functional p53 (representing more than half of the tumors), alternative anti-tumor therapies are under development based on induction of apoptosis independent of p53 (Thompson, 1995; Paulovich et al., 1997). For this, one has to search for the factors involved in induction of apoptosis that do not need p53 and/or cannot be blocked by anti-apoptotic activities, such as Bcl-2 or Bcr-ab1-like ones. These factors might be part of a distinct apoptosis pathway or might be (far) downstream of the apoptosis-inhibiting compounds.
Apoptin (also called “Vp3”, the terms being used interchangeably herein) is a small protein derived from chicken anemia virus (CAV; Noteborn and De Boer, 1996; Noteborn et al., 1991; Notebom et al., 1994; 1998a), which induces apoptosis in human malignant and transformed cell lines, but not in untransformed human cell cultures. In vitro and in vivo, Apoptin fails to induce apoptosis in normal lymphoid, dermal, epidermal, endothelial and smooth-muscle cells. However, when normal cells are transformed, they become susceptible to apoptosis by Apoptin. Long-term expression of Apoptin in normal human fibroblasts revealed that Apoptin has no toxic or transforming activity in these cells (Danen-van Oorschot, 1997; and Noteborn, 1996).
In normal cells, Apoptin was found predominantly in the cytoplasm, whereas in transformed or malignant cells, i.e., characterized by hyperplasia, metaplasia or dysplasia, it was located in the nucleus, suggesting that the localization of Apoptin is related to its activity (Danen-van Oorschot et al. 1997).
Apoptin-induced apoptosis occurs in the absence of functional p53 (Zhuang et al., 1995a) and cannot be blocked by Bcl-2, Bcr-ab1 (Zhuang et al., 1995), or the Bcl-2-associating protein BAG-1 (Danen-Van Oorschot, 1997a; Notebom, 1996).
Therefore, Apoptin is a therapeutic compound for the selective destruction of tumor cells, or other hyperplasia, metaplasia or dysplasia, especially for those tumor cells that have become resistant to (chemo-)therapeutic induction of apoptosis, due to the lack of functional p53 and (over-)expression of Bcl-2 and other apoptosis-inhibiting lesions (Noteborn and Pietersen, 1999). It appears that even pre-malignant, minimally transformed cells are sensitive to the death-inducing effect of Apoptin. In addition, Noteborn and Zhang (1998) have shown that Apoptin-induced apoptosis is suitable for the diagnosis and treatment of cancer-prone cells.
The fact that Apoptin does not induce apoptosis in normal human cells implies that there would be little or no toxic effect of Apoptin treatment in vivo. Noteborn and Pietersen (1998) and Pietersen et al. (1999) have provided evidence that adenovirus-expressed Apoptin does not have a toxic effect in vivo. In addition, in nude mice it was shown that Apoptin has a strong antitumor activity.